The c-Ab1 protein, originally identified as the cellular homolog of the v-ab1 oncogene product of Abelson murine leukemia virus (A-MuLV) (Goff et al, Cell 22:777 (1980); Wang et al, Cell 36:349 (1984)), is a tyrosine kinase of unknown function. Biochemical data suggest that c-Ab1 may regulate signal transduction events in the cytoplasm and processes in the nucleus. c-Ab1 is found primarily in the nucleus (Van Etten et al, Cell 58:669 (1989)), but it is also found in association with the plasma membrane and bound to actin filaments in the cytoplasm (Van Etten et al, Cell 58:669 (1989)); Van Etten et al, J. Cell. Biol. 124:325 (1994)).
The c-Ab1 protein has a complex structure that includes several domains common to proteins implicated in signal transduction pathways. Among these domains are the non-catalytic Src homology 2 and 3 (SH2 and SH3) domains and the tyrosine kinase (SH1) domain. SH2 and SH3 domains are modular components present in a large number of proteins (Pawson, Nature 373:573 (1995)). These domains are critical in the formation of stable signaling protein complexes, and have also been shown to regulate protein function (Feller et al, Trends Biochem. Sci. 19:453 (1994); Cohen et al, Cell 80:237 (1995); Pawson, Nature 373:573 (1995)). The SH3 domain suppresses the intrinsic transforming activity of c-Ab1 in vivo (Franz et al, EMBO J. 8:137 (1989); Jackson and Baltimore, EMBO J. 8:449-456 (1989)), while the SH2 domain is required for the transforming function of activated abl genes (Mayer et al, Mol. Cell. Biol. 12:609 (1992); Mayer and Baltimore, Mol. Cell. Bio. 14:2883 (1994)). The unique carboxy(C)-terminal region of c-Ab1, which is encoded by a single exon, contains several functional and structural domains that include a nuclear localization signal (Van Etten et al, Cell 58:669 (1989)), proline-rich sequences that have the potential to bind to SH3-domain-containing proteins (Feller et al, EMBO J. 13:2341 (1994); Feller et al, Trends Biochem. Sci. 19:453 (1994); Ren et al, Genes & Dev. 8:783 (1994)), a DNA-binding domain (Kipreos and Wang, Science 256:382 (1992)) and an actin-binding domain (Van Etten et al, J. Cell. Biol. 124:325 (1994); McWhirter and Wang, EMBO J. 12:1533 (1993)). Several serine/threonine residues within the C-terminal exon are phosphorylated by the cdc 2 kinase (Kipreos and Wang, Science 248:217 (1990)) and by protein kinase C (Pendergast et al, Mol. Cell. Biol. 7:4280 (1987)). The presence of multiple structural and functional domains within the c-Ab1 tyrosine kinase and its localization to cytoplasmic and nuclear cellular compartments, suggest a potential role for c-Ab1 in the regulation of transcription, DNA replication or cell cycle progression, as well as in the control of signaling events in the cytoplasm.
The tyrosine kinase activity of c-Ab1 is tightly regulated in vivo (Pendergast et al, Proc. Natl. Acad. Sci. USA 88:5927 (1991); Mayer and Baltimore, Mol. Cell. Bio. 14:2883 (1994)). Overexpression of c-Ab1 at levels 5- to 10-fold over the endogenous c-Ab1 protein does not lead to cell transformation but causes growth arrest (Jackson and Baltimore, EMBO J. 8:449 (1989); Jackson et al, EMBO J. 12:2809 (1993); Sawyers et al, Cell 77:121 (1994)). In contrast, structurally altered forms of Ab1 cause cell transformation and exhibit elevated tyrosine kinase activity when expressed at similar levels (Franz et al, EMBO J. 8:137 (1989); Jackson and Baltimore, EMBO J. 8:449 (1989); Muller et al, Mol. Cell. Biol. 11:1785 (1991)).
Activation of the oncogenic potential of c-Ab1 has been shown to occur as a consequence of structural alterations in the amino(N)- or C-terminal sequences (reviewed in Wang, Curr. Opin. Genet. Dev. 3:35-43 (1993)). Three naturally occurring c-ab1-derived oncogenes have been identified (Goff et al, Cell 22:777 (1980); Bergold et al, J. Virol. 61:1193 (1987); Pendergast and Witte, In: Balliere's Clinical Haematology 1(4):1001 (1987); Kurzrock et al, N. Engl. J. Med. 319:990 (1988)). Oncogenic activity has been shown to result from, or be associated with, deletion of the Ab1 SH3 domain and fusion with gag sequences following retroviral transduction (Franz et al, EMBO J. 8:137 (1989); Jackson and Baltimore, EMBO J. 8:449 (1989); Muller et al, Mol. Cell. Biol. 11:1785 (1991)), deletion of Ab1 C-terminal sequences and fusion with viral sequences, while retaining the Ab1 SH3 domain (Bergold et al, J. Virol. 61:1193-1202 (1987)), and fusion of bcr sequences upstream of the second exon of c-ab1 (Muller et al, Mol. Cell. Biol. 11:1785 (1991); McWhirter and Wang, Mol. Cell. Biol. 11:1553 (1991); Pendergast et al, Cell 66:161 (1991)). Mutants of c-Ab1 have also been generated experimentally that exhibit increased transforming activity. These include Ab1 proteins with deletions or alterations in the SH3 and C-terminal sequences (Franz et al, EMBO J. 8:137 (1989); Jackson and Baltimore, EMBO J. 8:449 (1989); Goga et al, Mol. Cell. Biol 13:4967 (1993); Mayer and Baltimore, Mol. Cell. Biol. 14:2883 (1994)). The structural alterations in the mutated Ab1 proteins disrupt the negative regulatory mechanisms that control the c-Ab1 protein tyrosine kinase, generating transforming Ab1 proteins that are constitutively active and are primarily localized in the cytoplasm.
Several possible mechanisms have been suggested for the inhibition of the c-Ab1 tyrosine kinase. Recently, it has been shown that the inhibitory effect of the Ab1 SH3 domain is extremely position sensitive (Mayer and Baltimore, Mol. Cell. Biol. 14:2883 (1994)). These results suggest that, in addition to the SH3 domain, other region(s) of c-Ab1 may be required for repression. Two potential mechanisms have been proposed. First, it is possible that the SH3 domain functions in cis by binding to another region of Ab1 and effectively locking the protein in an inactive conformation. A second model consistent with the available data suggests that the c-Ab1 protein is negatively regulated by a transacting cellular modulator that exerts its effects by interacting with the Ab1 SH3 domain and a second region of the Ab1 protein (Mayer and Baltimore, Mol. Cell. Bio. 14:2883-2894 (1994). The present invention provides such a protein.